The bacterium Vibrio cholerae is the cause of a lethal diarrheal disease called cholera. This organism commonly inhabits aquatic environments where it encounters unicellular eukaryotic `grazers' such as amoeba and ciliates which prey on bacterial cells. However, V. cholerae apparently has acquired gene clusters that provide some resistance to this predation and these genes may allow it to become a human pathogen. Two such groups of genes provide the blueprint for construction of Type III and Type VI secretion systems (T3SS and T6SS). These nanomachines allow V. cholerae puncturing amoeba and human cells and then injecting into them toxic proteins. Many other bacteria express T3SS and T6SS and thus they are exciting targets for development of drugs and vaccines. We have shown that T3SS and T6SS are important virulence factors in appropriate infection models for cholera but do not understand exactly how these systems enhance virulence properties such as intestinal colonization and fluid secretion responses. This proposal seeks funding for a research program to address the following general questions about the V. cholerae T3SS and T6SS: 1) Do T3SS effectors cause dynamic movements of host cell membranes in ways that provide an explanation for the pattern of adherence of V. cholerae to target cells? 2) Do injected effectors proteins of T3SS mediate adherence of V. cholerae to host cells by translocating a receptor that is recognized by a bacterial adhesin? 3) Does this translocated receptor transit through host cell by passing through subcellular compartments such as the ER and Golgi? 4) Can we define the mechanistic flexibility of the V. cholerae T6SS by proving that it can translocate heterologous proteins and effectors domains? 5) Can we understand the role of phagocytosis in the delivery of a toxic T6SS effector? 6) What is the mechanism of T6SS regulation and the roles of essential T6SS genes in the secretion and injection process? 7) Can we utilize highly parallel `systems biology' platforms such as protein chips and high-throughput genetics to define protein-protein interactions, novel immune activators and targets fo antimicrobial compound discovery?